Caliciviruses

John D Neill, National Animal Disease Center, Ames, Iowa, USA

The Caliciviridae is a family of small, nonenveloped viruses containing a single-stranded, plus-sense genomic RNA that is polyadenylated at its 3-end. Most caliciviruses have distinctive cup-shaped depressions (L. calix, cup) on their surface, giving them their characteristic Star of David appearance. These viruses have a large and diverse host range and cause diseases of both human and veterinary importance.

Secondary article
Article Contents
. Introduction . Classification . Structure . Replication . Epidemiology . Clinical Features . Control . Summary

Introduction
The caliciviruses were rst described with the outbreak of a virulent, highly contagious vesicular disease, later termed vesicular exanthema of swine (VES), in the United States in the early 1930s. The virus causing this disease, Vesicular exanthema of swine virus (VESV), was originally thought to be a foot-and-mouth disease virus (FMDV), but was later recognized as a novel virus because of a host range dierent from FMDV. San Miguel sea lion virus (SMSV) was rst isolated in 1972 from California sea lions on San Miguel Island, o the coast of California. This came about through investigations undertaken to nd the cause of large numbers of abortions and death losses in newborn sea lion pups. VESV and SMSV share morphological, immunological and genetic characteristics as well as causing vesicular diseases. Feline caliciviruses were initially isolated in the 1950s during attempts to isolate feline panleukopenia virus, a parvovirus. These viruses were described as rapidly cytopathic in cultured feline cells. Feline calicivirus (FCV) was later found to be associated with feline respiratory disease and is now considered a signicant feline pathogen around the world. Rabbit haemorrhagic disease virus (RHDV) was rst described in China in 1984 following an outbreak of acute viral haemorrhagic disease of rabbits. Since then, it has spread to other countries in Asia as well as Europe. European brown hare syndrome virus (EBHSV) was reported in Europe in 1986 but later studies of preserved liver samples from diseased hares place the possible incidence of the virus as early as 1976. The two viruses cause similar diseases but dier antigenically and in lagomorph genus infected. The human caliciviruses were described following immunoelectron microscopic analysis of clinical samples from a diarrhoeal outbreak of schoolchildren in Norwalk, Ohio in 1968. This virus, called the Norwalk agent, has since been determined to be one of a large group of caliciviruses causing gastroenteritis in humans. These viruses, termed small round structured viruses (SRSVs), do not have the characteristic cup-shaped depressions and were not initially classied as caliciviruses. A second group,

the classical human caliciviruses (HuCVs), which possess the classical calicivirus morphology by electron microscopy, causes diarrhoeal disease essentially identical to that caused by the SRSVs.

Classification
A very recent change by the International Congress on the Taxonomy of Viruses (ICTV) in classication has divided the Caliciviridae into four genera, the Lagovirus, Vesivirus, Norwalk-like viruses and Sapporo-like viruses (Pringle, 1998). A detailed phylogenetic study supporting this classication was done by Berke et al. (1997), conrming the genus assignments. FCV, SMSV and VESV were initially classied as a conditional genus of the Picornaviridae. They were later given their own family designation with a single genus, because they were morphologically dierent from the picornaviruses, composed of a single structural protein and produced a subgenomic ribonucleic acid (RNA). Subsequent molecular biological characterization has shown that, in addition to the above dierences, the caliciviruses possess a longer genomic RNA with multiple open reading frames (ORFs) and encode the nonstructural proteins in the 5-end and the structural protein in the 3-end of the genomic RNA, the opposite of the picornaviruses. Considering these dierences, it is interesting to note that identication of three of the nonstructural proteins of the caliciviruses was made based on identication of conserved amino acid motifs in common with nonstructural proteins encoded by picornaviruses. RHDV and EBHSV are the two species placed in the Lagovirus genus. Lagovirus host range is limited to lagomorphs, or, specically, the European rabbit (Oryctolagus cuniculus) and hares (Lepus europaeus and L. timidus). These viruses are classied in this genus because of the rabbit or hare host, single large ORF encoding both the nonstructural and structural proteins and similarity in disease syndrome (Table 1). The two species are distinguishable antigenically and show only 6369% similarity at the
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Table 1 Comparison of properties of caliciviruses Lagovirus Host range Disease Rabbits, hares Haemorrhagic disease; hepatitis Yes No 7.35 2 2345 Yes 60 Vesivirus Broad many species Vesicular lesions; abortions; upper respiratory infections Yes Yes 7.78.3 3 17631878 Yes 7378a 5961b Norwalk-like Humans Diarrhoea Sapporo-like Humans Diarrhoea

Typical calicivirus morphology Grow in culture Size of genome (kb) Number of ORFs Length of ORF1 (aa) Subgenomic RNA Size of capsid protein (kDa)

No No 7.65 3 1790 ? 5860c

Yes No 7.4 2 2281 ? 5860c

aa, amino acids; kDa, kilodaltons; ORF, open reading frame. ?, unknown but inferred to produce a subgenomic RNA based on similarities to other caliciviruses. a Capsid precursor protein. b Mature capsid protein. c Calculated from amino acid sequence.

amino acid level in capsid protein sequences. The type species is RHDV. FCV, SMSV and VESV belong to the Vesivirus genus, named for the vesicular disease caused by many of these viruses. The two species comprising the Vesivirus genus are FCV and VESV (the type species). The VESV species encompasses all VESV and SMSV strains as well as several viruses that were not placed into either of these two groups. These include Primate calicivirus (Pan-1), Bovine calicivirus (Bos-1), Cetacean calicivirus (Tur-1) and Reptile calicivirus (Cro-1). Biochemical characterization has shown that VESV and SMSV are indistinguishable morphologically and immunologically. Both groups cause similar vesicular disease in both pinniped and porcine species. Recent nucleotide sequence comparisons have demonstrated that these two groups of viruses comprise a single genogroup within the Caliciviridae. The Vesivirus genome contains three ORFs and has distinct frames to encode the structural and the nonstructural proteins. Members of this genus are the only caliciviruses that can be propagated in cell culture (Table 1). The human caliciviruses were previously placed into one of two groups called the small round structured viruses (SRSVs) or the classical human caliciviruses (HuCVs). The SRSVs, which include Norwalk virus and the Snow Mountain and Hawaii agents, do not show the typical calicivirus morphology of well-dened cups on their surface by negative-stain electron microscopy, but rather have a fuzzy, ill-dened border. Thus, their classication was uncertain. Recent molecular biological analysis has shown that the SRSVs are caliciviruses, based on genome
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structure and conservation of functional amino acid motifs in both capsid and nonstructural proteins. The SRSVs encode the nonstructural and capsid protein in separate ORFs. The classication of the HuCVs, which include the Sapporo and Parker viruses, was more clear-cut because of their characteristic calicivirus morphology. The ORFs encoding the capsid and nonstructural proteins are fused into one reading frame, much like that of the lagoviruses, as opposed to two frames in the SRSVs. Analysis of viral nonstructural protein amino acid sequences showed a closer relationship to the animal caliciviruses than to the SRSVs. The HuCVs are also antigenically distinct from the SRSVs. Based on these data, these viruses have been assigned to the Norwalk-like (SRSV) and the Sapporolike (HuCV) genera. Until recently, the viral agent causing non-A, non-B hepatitis in humans, Hepatitis E virus (HEV), was classied as a conditional member of the Caliciviridae, based on apparent similarity between HEV and caliciviruses by negative-stain electron microscopy and similarities in genome organization. The Calicivirus Study Group submitted a proposal to the ICTV recommending the removal of HEV from the Caliciviridae into an unclassied status. This was done on the basis of lack of phylogenetic relatedness of HEV to the caliciviruses as well as substantial dierences in the nonstructural proteins encoded by the viruses. The classication of HEV still remains to be determined. A summary and comparison of the properties of the caliciviruses can be found in Table 1.

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Structure
Virion
Calicivirus virions are nonenveloped and have T 5 3 icosahedral symmetry. The virus particles are 2740 nm in diameter based on negative stain electron microscopy. The buoyant density of caliciviruses ranges from 1.33 to 1.41 g mL 2 1 in caesium chloride gradients. The virus coat is composed of 180 copies of the single capsid protein with 32 cup-shaped depressions on the surface of the virus particle found at the ve-fold and three-fold axes of symmetry. These depressions are readily visible in most caliciviruses (Figure 1a, b) but may be faint to nonexistent in others. A more detailed analysis of the virion structure was conducted with Primate calicivirus (Pan-1), a member of the Vesivirus genus. Using electron cryomicroscopy and computer imaging of highly puried virus, a threedimensional structure at approximately 2.2-nm resolution was derived. The virus particles were 40.5 nm in diameter. They contained 32 large surface hollows that were 5 nm deep and 9 nm wide. The capsid proteins were grouped in 90 dimers or capsomeres that formed arches surrounding the cup-like depressions. Each capsomere was composed of three distinct domains: the S, found at the base of the arches that probably forms the shell of the virus; the P1, a bilobed distal end forming the top of the arch; and P2, the stem of the arch. Determination of which amino acids make up these three domains awaits the solving of the highresolution three-dimensional structure of a calicivirus.

Viral genome
The genome of the caliciviruses consists of a single RNA molecule ranging from 7.4 to 8.3 kb in length. The genomic RNA of the caliciviruses contains 23 ORFs (depending on the genus) that encode the structural (capsid) and the nonstructural proteins (Figure 2). The nonstructural ORF is separate from the structural ORF in the vesiviruses and the Norwalk-like viruses but the two frames are fused into a single ORF in the lagoviruses and the Sapporo-like viruses. The subgenomic RNAs range from 2.2 to 2.7 kb in length and have been demonstrated in vesivirus and lagovirus-infected cells. The subgenomic RNA is the major template for translation of the single capsid protein encoded by the second ORF found in the 3-end of the genomic RNA. The genomic RNA as well as the subgenomic RNA has a small protein (VPg) covalently attached to the 5-end, while the 3-end of these RNAs is polyadenylated. All caliciviruses examined thus far have a small ORF at the 3-end of the genomic RNA that encodes a small, basic protein of unknown function. The end of the capsid protein ORF overlaps at least the initiation codon of this small ORF. There is evidence in FCV that the 3-ORF is translated from the same subgenomic RNA as the capsid protein, perhaps by a ribosomal frameshift mechanism.
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Figure 1 Electron micrographs of caliciviruses. (a) Negative-stain electron micrograph of San Miguel sea lion virus serotype 4 following caesium chloride isopycnic centrifugation of virus grown in Vero cells. Bar, 57 nm. (b) Negative-stain electron micrograph of Feline calicivirus following caesium chloride isopycnic centrifugation of virus grown in Crandell Rees feline kidney (CRFK) cells. Bar, 67 nm. (c) Thin-section electron micrograph of aggregate of Feline calicivirus in an infected CRFK cell. Note nucleus and swollen nuclear membrane in upper left hand corner. Bar, 200 nm.

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Caliciviruses

ORF2 Helicase VPg Protease Polymerase Capsid

VPg Lagovirus

7.35 kb Subgenomic RNA VPg 2.1 kb

Capsid Helicase VPg Protease Polymerase ORF3

Poly(A) Poly(A)

VPg Vesivirus

FCV 7.7 kb

SMSV/VESV 8.3 kb VPg 2.42.7 kb

Poly(A) Poly(A)

Subgenomic RNA

Capsid Helicase VPg Protease Polymerase ORF3

VPg Norwalk-like viruses

7.65 kb

Poly(A)

ORF2 Helicase VPg Protease Polymerase Capsid

VPg Sapporo-like viruses

7.4 kb

Poly(A)

Figure 2 Comparisons of genomic RNAs from the Lagovirus, Vesivirus, Norwalk-like viruses and Sapporo-like viruses genera. The single line represents the genomic RNA with the genome-linked viral protein (VPg) at the 5-end and the poly(A) tail at the 3-end. The subgenomic RNAs that have been characterized are shown beneath the region for which they are equivalent in sequence. The sizes of the RNAs in kilobases (kb) are shown. The boxes above the genomic RNA represent the ORFs in different reading frames with the relative position of the encoded and identified proteins. FCV, Feline calicivirus; SMSV, San Miguel sea lion virus; VESV, Vesicular exanthema of swine virus. The figure is not drawn to scale.

Viral proteins
The nonstructural proteins are encoded in a large ORF beginning near the 5-end of the genomic RNA and are synthesized as a single large polyprotein. Posttranslational proteolytic cleavage of the polyprotein by a viral cysteine protease found near the C-terminus of the polyprotein has been demonstrated in RHDV, FCV and Southampton virus, a member of the Norwalk-like genus. The processing by this viral protease begins with the autocatalytic cleavage of its N-terminal border within the polyprotein, followed by processing of the remainder of the polyprotein to yield the mature proteins. Other identied proteins derived from the nonstructural polyprotein include an RNA helicase, VPg and an RNA-dependent RNA polymerase (Figure 2). The order of the nonstructural proteins within the polyprotein is similar to that found in the picornaviruses; however, the picornaviruses encode the nonstructural proteins downstream of the structural proteins, the opposite of the caliciviruses. The single capsid protein ranges from 58 to 61 kDa in size (Table 1). This protein is translated as a larger capsid precursor protein in the vesiviruses, with the N-terminal prodomain being posttranslationally cleaved at some point following synthesis. This capsid protein cleavage is also
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mediated by the viral cysteine protease. In the remaining genera, the capsid protein is synthesized in its mature form. The capsid protein contains a highly conserved region that forms a b-barrel domain that probably forms the internal shell of the virus particle. Also identied in the capsid protein are highly divergent or hypervariable domains that are believed to be exposed on the surface of the virus particle and contain the major antigenic determinants of the virus. Amino acid changes in these domains may cause changes in antigenicity, allowing persistence in the presence of immune surveillance. The small 1215-kDa protein encoded by the small 3ORF (ORF2 or ORF3 in Figure 2) serves an unknown function. It contains a high percentage of conserved basic residues and it may play a role in binding viral RNA and encapsidation. The lagovirus virion contains a 1015-kDa protein as a minor component that was shown to be the product of the 3-ORF.

Replication
Little is known concerning the steps in viral replication. The viral receptor has not yet been identied, but

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preliminary data from studies using FCV indicate that it contains polysaccharides that are important in recognition and/or binding by the virus. An acidic environment is necessary for uncoating of the virus particle following internalization. Replication of the viruses takes place in the cytoplasm and does not require nuclear involvement. Viral replication occurs in the presence of actinomycin D (an inhibitor of RNA synthesis from a double-stranded deoxyribonucleic acid (DNA) template) or in enucleated cells. Late in infection, there is a proliferation of vacuoles and membranes within the infected cell that appear to have virus particles lined along the cytoplasmic side. It is possible to nd aggregates of virus within the cytoplasm (Figure 1c). Newly synthesized viral RNA is detectable in infected cells as early as 2 h after infection. The FCV subgenomic RNA is detected at 2 h after infection, with transcription reaching steady state levels at 45 hours. Synthesis of genomic plus-strand RNA is detectable at 4 h after infection. The genomic RNA is replicated from a fulllength negative-strand copy that is readily detected at 3 h after infection. These results demonstrate that production of the subgenomic RNA and negative-strand genomic RNAs occur rst and are probably linked, with the subgenomic RNA being transcribed from the genomic negative strand. The 5-untranslated regions of the genomic and subgenomic RNAs of the caliciviruses share a high degree of sequence similarity, indicating that the complementary 3-terminal and internal sequences of the negative-strand RNA may be specically recognized by the viral replicative/transcriptional machinery (Figure 3). A double-stranded RNA corresponding in size to the subgenomic RNA has been reported; however, it is present at very low levels and is not believed to play a role in replication. Other minor viral RNA species with no known function have also been detected in FCV-infected cells. The genomic plus-strand RNA acts as messenger RNA after virion uncoating and release of the RNA into the cytoplasm of the infected cell. The nonstructural ORF is translated rst for the synthesis of proteins necessary for RNA replication and transcription. The nonstructural polyprotein is posttranslationally processed by a viral cysteine protease, as mentioned above. The cysteine protease may only poorly cleave itself from the RNAdependent RNA polymerase, resulting in a single protein that may contain both functions. Alternatively, from analogy to the picornaviral 3CD protein, this inecient cleavage may represent a regulatory mechanism to limit the concentration or activity of the RNA polymerase, while not compromising protease activity. Further work is necessary to determine this. Viral proteins are detected as early as 3 h after infection in FCV-infected cells and continue to accumulate until at least 8 h after infection (Figure 4). The mature capsid protein is present as a major band at 60 kDa. There are at least ve nonstructural proteins in FCV-infected cells based on in vitro translation

1G

T A A A A G A A A T T T G A G A C A A T G T C T25 : : : : : : : : : : : : : : : : : : T G T T C G A A G T T T G A G C A T G TG C T5321

5297G

(a)
1G

T A A A T G A G A A T T T G A G C T A T GG C T25 : : : : : : : : : : : : : : : : : : : T T G A G A A T T A G C C A C T A T GG C T5673

5650G T

(b)
1G

T G A A A G T T A T G G C GG C T A T G T CG24 : : : : : : : : : : : : : : : : G A A T G T T A T G G A G G G C A A A G C C5319

5296G T

(c)

1G

T G A A T G A T G A G G C G T C G A A A G A23 : : : : : : : : : : : : : : : : : : : A A A T G A T G A T G G C G T C T A A G G A5374

5351G T

(d)
Figure 3 Sequence comparison between the 5-ends of the genomic and subgenomic RNAs of (a) Feline calicivirus (FCV); (b) San Miguel sea lion virus (SMSV); (c) Rabbit haemorrhagic disease virus (RHDV); and (d) Norwalk-like viruses. The 5-terminal bases are aligned, with identical bases marked with a colon (:) and missing nucleotides indicated by a hyphen. The comparisons are FCV (CFI strain); SMSV serotype 1; RHDV and Norwalklike (Southampton virus). The nucleotide numbers from the 5-end of the genomic RNA are indicated. The ATG initiation codon is underlined, with multiple ATGs indicated in the Norwalk-like sequences because the authentic start of translation is unknown.

Figure 4 Western immunoblot analysis of proteins extracted from Feline calicivirus (FCV)-infected Crandell Rees feline kidney (CRFK) cells at various times after infection. The cells were collected at the time points indicated above each lane (hours postinfection) and the proteins were extracted. The lane marked C (control) represents uninfected CRFK cells. Following electrophoresis of proteins on a sodium dodecyl sulfatepolyacrylamide gel, the proteins were blotted to nitrocellulose and probed with FCV hyperimmune serum collected from a cat that had been infected with FCV. The capsid protein is the major protein band at approximately 60 kDa. The numbers on the left represent the size markers in kilodaltons.

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and processing studies. Figure 4 shows more than ve viral proteins in infected cells. It is not certain which are the mature proteins and which may represent processing intermediates. FCV, RHDV and Southampton virus possess cysteine protease activity, as demonstrated by processing of the nonstructural polyprotein in vitro.

Epidemiology
FCV is a ubiquitous viral pathogen of numerous feline species. It is common in domesticated cats and has been isolated from cheetahs in Australia, as well as several feline species in zoos. While these viruses are readily transferred from animals with an active infection, the major source of virus in feline populations is the chronic, subclinical carrier. The carrier state follows the acute phase of the disease, and plays an important role in maintaining these viruses in the feline population. Chronically infected cats shed virus from sites of low-level replication in the tonsils and surrounding tissues. Small numbers of virus in oral secretions readily infect naive contacts. This low-level replication and shedding occurs despite the presence of a protective antibody response. Recent ndings show that antigenic variants of the initial infecting virus can be recovered from chronically infected cats. This indicates that generation of heterogeneity during replication, a trait common to RNA viruses, plays an important role in establishment and maintenance of chronic infections. SMSV has been isolated from many pinniped and cetacean species as well as marine sh. Antibodies against a number of SMSV as well as VESV serotypes have been found in numerous cetaceans and pinnipeds. It has been proposed that the marine mammal population may be a reservoir from which the viruses may become re-established in domestic livestock herds. Both SMSV and VESV have been shown to infect domestic animal species such as pigs and cattle. SMSV is propagated in cell culture and are routinely grown in cells of primate origin as well as having been isolated from primates. Other viruses that belong to the Vesivirus genus, Pan-1, Bos-1, Tor-1 and Cro-1, illustrate the diverse species that can be infected. Recently, it was shown that SMSV caused a zoonotic infection of a laboratory worker, adding humans to the host range. VESV is spread by direct contact with infected animals as well as from feeding of raw garbage containing meat scraps from infected animals. VES rst appeared in the early 1930s as widespread, seemingly unrelated outbreaks. The only common factor was the feeding of uncooked garbage from restaurants that served seafood. Later outbreaks were associated with the feeding of raw pork scraps to infected herds. These outbreaks were essentially limited to California until 1951, when a train loaded with fresh pork carcasses headed west from San Francisco, California and left raw pork trimmings in Wyoming, which
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were fed to swine. This began an epidemic that did not end until late 1956 after it became compulsory for all garbage fed to pigs to be cooked. The disease was ocially declared exotic in the United States in 1959 after 3 years of no conrmed cases. The lagovirus RHDV was rst reported in China in 1984 in an outbreak associated with the importation of rabbits from Germany. Outbreaks were soon reported from other countries, and were thought to be associated with rabbit meat imported from China. However, RHDV was recognized in Europe in domestic rabbits at the same time it was observed in wild rabbit populations, indicating that it was already present. The virus appeared in Mexico in the late 1980s and was quickly eradicated by elimination of infected animals. EBHSV was recognized earlier in Europe and may have been present as early as 1976. These viruses are spread by oral, nasal and parenteral transmission. The virus is present in all secretions of infected animals. It is not clear if insect vectors play a role in transmission, although it appears to have been the means of escape of RHDV from Wardang Island, o the coast of southern Australia, where it was being tested as a biological control agent for European rabbits. Seasonal insects borne on the wind appear to have carried it to the mainland, where it infected rabbits and has since spread to much of Australia. The spread of the virus has resulted in the death of 6590% of the rabbits in some areas. The human caliciviruses are recognized as a major cause of gastroenteritis, with the main means of transmission of the viruses being by the oralfaecal route. The sources of the viruses are many but major sources in foodborne disease are shellsh harvested in sewage-containing waters and foodstus contaminated by infected workers. Many of the cases in an outbreak are secondary and are not related directly to the original source of the outbreak. Norwalklike and Sapporo-like viruses produce essentially identical clinical symptoms in infected individuals; however, they dier in their epidemiology and immunology. The Norwalk-like viruses are primarily associated with disease in school-aged children and adults and the Sapporo-like viruses are found primarily in cases involving infants and young children. The Sapporo-like viruses will produce immunity that may prevent later reinfection. The Norwalk-like viruses produce a shortlived immunity. This disappears after a short time; the individual may be reinfected.

Clinical Features
FCV infections in cats display a wide range of clinical symptoms. The infections may be subclinical or acute, characterized by rhinitis, tracheitis and pneumonia, as well as vesicle formation followed by ulceration of the oral epithelium. Accompanying these may be fever, anorexia

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and ocular and nasal discharges. A transient limping syndrome has also been reported, characterized by muscle soreness and joint stiness in kittens. Mortality can reach 30% in animals less than 12 weeks of age, and is usually caused by extensive lung consolidation associated with pneumonia. A viral carrier state is often established following recovery from disease. These are cats that have been infected with FCV and, after recovery, continue to shed the virus at low levels. Replication is within the tonsils and surrounding tissues. Infection of swine by VESV causes a rise in body temperature (1051068F; 40.5418C), followed by formation of vesicles on the snout, lips, tongue and oral mucosa. The primary vesicles rupture after 4872 h, leaving a red, raw wound. This causes the spread of virus contained in the liberated vesicular uid to surrounding tissues, particularly the oral mucosa, soles, interdigital space and coronary bands of the feet. The appearance of secondary vesicles on the feet causes great pain in walking, and the animal may refuse to walk until the lesions are healed. The clinical features caused by SMSV are very similar to those of VESV. Marine mammals infected with SMSV display vesicle formation on the cooler portions of the body, such as ippers. Infection with SMSV has also been associated with reproductive failure and death of newborn pups. Infection of swine with SMSV causes disease indistinguishable from that caused by VESV. The acute disease caused by RHDV infection in the European rabbit is characterized by anorexia and tachypnoea, convulsions and occasional bloody nasal secretions. Fever is common early in infection, but low body temperature is observed just before death. Experimental infections usually cause death in 4872 h. Internally, most of the major organs are aected in rabbit haemorrhagic disease. The lungs, trachea and kidneys show haemorrhages, while the liver and spleen are enlarged and show a dark reddish discoloration. Death is the result of necrotizing viral hepatitis. Disseminated intravascular coagulation may be observed. Rabbits less than 4 weeks of age do not display clinical signs and generally survive infection. Hares infected with EBHSV show disease symptoms similar to RHDV. EBHSV infections show essentially the same pathogenesis, clinical appearance and mortality rate as those of RHDV. Clinically, infection by human caliciviruses is seen as acute and self-limiting, with symptoms of vomiting, diarrhoea, abdominal cramping and fever lasting 12 24 h. The average incubation period is 48 h. Estimates place the number of people exposed that actually become ill from human calicivirus infection at 50%. Hospitalization is rarely required, with the major impact from infection being time lost from school and work. Diagnosis of human calicivirus infection is at best dicult and is based on clinical examination. Diagnoses of infection in outbreaks can be made with the following criteria: (1) absence of bacterial or parasitic pathogens; (2) vomiting in more than

50% of cases; (3) duration of illness from 12 to 60 h; and (4) incubation period of 2448 h (Estes and Hardy, 1995). Owing to the inability to propagate these agents in cell culture systems, other diagnostic tests were developed. The most common test, immunoelectron microscopy, employs a patients pre- and postinfection serum to aggregate dierentially virus present in a collected stool specimen. With the advent of laboratory-produced antigen in the form of recombinant baculovirus-synthesized virus-like particles, immunological tests, such as enzyme-linked immunosorbant assays (ELISAs), have been developed. This has resulted in greater insight into the immune response and virus shedding pattern of infected individuals.

Control
Vaccination for calicivirus infection, as with other viruses, will probably remain the single most important means of control. Unfortunately, vaccines are not currently available for all caliciviruses. A vaccine for FCV has been available for some time, utilizing live, avirulent strains. The most commonly used vaccine virus is the F9 strain, which was shown to be approximately 50% protective against a number of eld viruses. In another study, FCV strains isolated from previously vaccinated cats showed little or no resemblance to the vaccine strain, indicating that a monovalent FCV vaccine is unlikely to protect against all eld viruses, thus resulting in vaccine breaks. A quarantine and eradication programme was used from 1932 to 1954 to control the spread of VESV. Stopping the movement of swine from infected herds initially met with limited success; however, it became apparent it was insucient to stop the spread of the disease. Control and eradication of VESV was achieved in the late 1950s in the United States by requiring that all garbage fed to domestic pigs be cooked. VES is now considered an exotic disease in the United States. There is no control programme in place to control SMSV, as it is currently considered a disease of marine mammals and not a problem in domestic livestock herds; however, antibodies against VESV and SMSV have been detected in the sera from domestic cattle and swine herds in recent studies. Control of the disease caused by lagoviruses is more dicult because of the presence of the viruses in wild rabbits and hares. Inactivated vaccines are used in farmed populations and have been eective in preventing disease. Several recent reports describe the use of virus-like particles (VLPs), produced in recombinant baculovirusinfected insect cells, to be eective as vaccines. Strict guidelines have been implemented to prevent the introduction of RHDV and EBHSV into commercial operations. Serological evaluation is done to conrm that the animal is
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seronegative. It is then placed into quarantine for 2 weeks along with a naive contact rabbit before entry into the general population. Contact with wild animals is avoided, as well as with rabbits from noncommercial sources. Workers must use a high level of hygiene, with change of clothes and showering. Animals that are diseased or those assumed to be infected are destroyed. It is not clear whether immunization will be eective in prevention of illness caused by the human caliciviruses. Currently, there is no vaccine available. From studies examining the immune response to these viruses, shortterm immunity may be elicited following an infection that increases resistance to reinfection, but long-term protective immunity is questionable. Preliminary trials have been conducted using the VLPs produced from overexpression of the capsid protein gene of Norwalk virus in a baculovirus expression system. These particles were shown to be safe as well as immunogenic in volunteers, but the ability of these particles to confer protective immunity still remains to be determined. The current means of control is the complete cooking of all shellsh and the monitoring of waters where shellsh are harvested for contamination by sewage. The health of food workers is also monitored, as is maintenance of good hygienic practices to prevent faecaloral transmission from infected workers.

availability of a relatively eective vaccine for FCV, and the inability to propagate many of these viruses in cell culture. As these viruses have become associated with more disease states, interest in them has increased, especially in dening their molecular biology. Recent innovative approaches, such as production of VLPs for RHDV and the human enteric caliciviruses, will allow greater analysis of the immunological response to infection as well as the development of eective vaccines to control disease caused by caliciviruses.

References
Berke T, Golding B, Jiang X et al. (1997) Phylogenetic analysis of the caliciviruses. Journal of Medical Virology 52: 419424. Estes MK and Hardy ME (1995) Norwalk virus and other enteric caliciviruses. In: Blaser MJ, Smith PD, Ravdin JI, Greenburg HB and Guerrant RL (eds) Infection of the Gastrointestinal Tract, pp. 1009 1034. New York: Raven Press. Pringle CR (1998) Virus taxonomy San Diego 1998. Archives of Virology 143: 14491459.

Further Reading
Clarke IN and Lambden PR (1997) The molecular biology of the caliciviruses. Journal of General Virology 78: 291301. Du JP, Chasey D, Munro R and Woolridge M (1994) European brown hare syndrome in England. Veterinary Record 134: 669673. Green KY (1997) The role of human caliciviruses in epidemic gastroenteritis. Archives of Virology. Supplementum 13: 153165. Prasad BVV, Matson DO and Smith AW (1994) Three-dimensional structure of caliciviruses. Journal of Molecular Biology 240: 256264. Smith AW, Skilling DE, Barlough JE and Berry ES (1986) Distribution in the north Pacic Ocean, Bering Sea and Arctic Ocean of animal populations known to carry pathogenic caliciviruses. Diseases of Aquatic Organisms 2: 7386.

Summary
The caliciviruses are a family of viruses that have a large host range and cause a broad spectrum of diseases. These viruses have been known for some time, but relatively little has been done to characterize them in greater detail because of the eradication of VESV in the late 1950s,

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